Ultrapure hypoallergenic solutions of sacrosidase

ABSTRACT

One aspect provides an ultrapure, hypoallergenic sacrosidase. Another aspect provides a solution of sacrosidase in about 1:1 glycerol/water having an enzymatic activity of at least about 7500 IU/mL and a residual papain concentration that does not include an allergic reaction in a human patient when given a dose of about 2.0 mL/day.

CLAIM OF PRIORITY

This application is a continuation and claims the benefit of priority ofU.S. patent application Ser. No. 15/270,970, filed on Sep. 20, 2016,which is a continuation and claims the benefit of priority to U.S.patent application Ser. No. 14/828,006, filed on Aug. 17, 2015 whichclaims the benefit of priority of U.S. patent application Ser. No.14/175,263, filed on Feb. 7, 2014, which are incorporated by referenceherein in their entirety.

BACKGROUND OF THE INVENTION

Congenital sucrose-isomaltase deficiency (CSID) is a chronic, autosomalrecessive, inherited, phenotypically heterogenous disease with variableenzyme activity. CSID is usually characterized by a subject havingcomplete or almost complete lack of endogenous human sucrase activity,along with a very marked reduction in isomaltase activity, a moderatedecrease in maltase activity, and the subject can have normal orabnormal lactase levels.

The human enzyme sucrase-isomaltase is naturally produced in the brushborder of the small intestine, primarily the distal duodenum andjejunum. The natural human enzyme hydrolyzes the disaccharide sucroseinto its component monosaccharides, glucose and fructose. Isomaltasebreaks down disaccharides from starch into simple sugars.

In the absence of endogenous human sucrase-isomaltase enzyme, as inCSID, sucrose is not metabolized. Unhydrolyzed sucrose and starch arenot absorbed from the intestine and their presence in the intestinallumen leads to osmotic retention of water. This may result in loosestools or diarrhea. Unabsorbed sucrose in the colon is fermented bybacterial flora to produce increased amounts of hydrogen, methane andwater. As a consequence, excessive gas, bloating, abdominal cramps,nausea, vomiting, and explosive diarrhea may occur. Chronicmalabsorption of disaccharides may result in malnutrition.Undiagnosed/untreated CSID patients often fail to thrive and fall behindin their expected growth and development curves. Prior to the FDAapproval of the commercial replacement product Sucraid® (sacrosidase)Oral Solution, the treatment of CSID has required the continued use of astrict sucrose-free diet with limited success in disease management.

CSID is currently treated by the oral administration, with meals, of aglycerol-water (1:1 w/w) solution of sacrosidase, which provides theenzyme replacement therapy for CSID. This solution is commerciallyprovided as Sucraid® (sacrosidase) Oral Solution distributed by QOLMedical LLC. Each milliliter (mL) of Sucraid® contains 8500International Units (I.U.) of the enzyme sacrosidase, the activeingredient. The chemical name of this enzyme is β,D-fructofuranosidefructohydrolase. The enzyme is derived from baker's yeast (Saccharomycescerevisiae), by enzymatic digestion with papain.

The Sucraid® product was typical of the era with respect to purity. TheFDA established purity specification was not less than 85% in the mainband(s) by reduced SDS-PAGE densitometry analysis following Coomassiestaining. This is a band ratio of sacrosidase to other proteins of about6:1.

The purity was specified by the FDA to comprise not more than 10 μg/mLof papain in the sacrosidase drug substance. The “drug substance” is theprecursor glycerol:water solution of sacrosidase that can be furtherdiluted and packaged to yield Sucraid®, which is referred to as thefinished “drug product.” The original label approved by FDA in 1998 madenotice of potential for allergic reactions, and cautioned doctors totreat CSID patients for the first time within their unit in caseallergic reactions arose. Again, in 2008, as the result of a site changeof the drug substance manufacturer, the FDA only approved the re-launchof this drug with the imposition of a Risk Evaluation and MitigationStrategy (REMS) which are only required by the FDA for drugs with thepotential for serious safety problems.

It has been reported that the primary structure of sacrosidase consistsof 513 amino acids with an apparent molecular weight of 100,000 g/molefor the glycosolated monomer (range 60,000-116,000 g/mole). Reports alsosuggest that the protein exists in solution as a monomer, dimer,tetramer, and octomer ranging from 100,000 g/mole to 800,000 g/mole. Ithas an isoelectric point of 4.1 (pI=4.093).

Presently, Sucraid® is provided in bottles containing 118 mL of thesacrosidase solution. A typical dose is either 1 or 2 mL with every mealor snack. The solution is bottled aseptically; however, it may becomecontaminated after opening due to the necessity of frequentadministration, so patients are instructed to discard the bottle 4 weeksafter opening.

SUMMARY OF THE INVENTION

This invention provides an ultrapure hypoallergic sacrosidase that meetsthe pharmaceutical standards for human prescription use. The sacrosidaseis also organoleptically improved, since aqueous solutions thereof areodorless.

One embodiment provides a protein composition consisting of sacrosidaseand containing no other proteins as determined by SDS-PAGE of up toabout 15 μg of said composition. Techniques of SDS-PAGE, as describedherein below, can employ reducing conditions and Coomassie BlueStaining. Thus, one embodiment provides a protein composition consistingessentially of sacrosidase having a band volume ratio of sacrosidase toother proteins of at least about 35:1, e.g., about 35-55:1, asdetermined by SDS-PAGE of about 20 μg of said composition. The proteincomposition of one embodiment is produced by a process comprisingdiafiltration of the yeast digest and does not employ chromatographicpurification of said sacrosidase.

Another embodiment provides a drug substance and a drug productconsisting essentially of a solution of sacrosidase in water comprisingabout 45-54 wt-% glycerol and that contains less than about 10 ng/mL,preferably less than about 3.0 ng/mL of papain in combination with atleast about 7500 IU/mL of sacrosidase. One embodiment provides asolution consisting essentially of at least about 7500-9500 IU/mL,preferably about 8500 IU/mL sacrosidase in about 1:1 glycerol:water thatcontains less than about 3.0 ng/mL papain. This sacrosidase formulationdoes not induce an allergic reaction in a human subject such as a CSIDpatient, when administered at a dose of up to about 2.0 mL (daily dosesof 2-10 mL).

Another embodiment provides a sacrosidase solution in water/glycerolhaving an activity of at least about 10,000 IU/mL and having less thanabout 10 ng/mL residual papain. In one embodiment, the solution hasabout a 1:1 wt-wt ratio of water to glycerol. In another embodiment, thesolution has a pH of about 4.1-4.5.

The amount of papain can be determined by the techniques of ELISA. Thesolution is also not produced by a process comprising chromatographicpurification of said sacrosidase.

As used herein the term “about” is intended to encompass variation inthe referenced parameter, such as concentration, pH, enzymatic activity,time and the like that would be recognized by the art worker asnecessarily inherent in determination of the parameter, see FIG. 1.

Additional information about Sucraid® and sacrosidase can be found inU.S. patent application Ser. No. 13/786,956, filed Mar. 6, 2013, whichis incorporated by reference herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow diagram of the current sacrosidase drug substancemanufacturing process.

FIG. 2. Optimal Loaded SDS-PAGE gel Stained with Coomassie Blue.Sacrosidase bulk drug substance (FIG. 1) from two manufacturers (5 μgprotein). Lane 1: Low range MW marker 17, 28, 34, 40, 73 and 102 kD;Lane 2: Reference Standard C4546; Lane 3: STS-199NS; Lane 4: STS-220NS;Lane 5: STS-241NS; Lane 6: 125275; Lane 7: 125434; Lane 8: 125722; Lane9: Reference Standard C3955; Lane 10: Broad range MW marker (21, 34, 45,66, 97 kD and 200 kD).

FIG. 3. Overloaded SDS-PAGE gel Stained with Coomassie. Sacrosidase bulkdrug substance from two manufacturers (20 μg protein). Lane 1: Low rangeMW marker; Lane 2: Reference Standard C4546; Lane 3: STS-199NS; Lane 4:STS-220NS; Lane 5: STS-241NS; Lane 6: 125275; Lane 7: 125434; Lane 8:125722; Lane 9: Reference Standard 03955; Lane 10: Broad range MWmarker.

FIG. 4. Overloaded Native gel stained with Coomassie. Sacrosidase bulkdrug substance from two manufacturers (15 μg protein). Lane 1: ReferenceStandard C4546; Lane 2: STS-199NS; Lane 3: STS-220NS; Lane 4: STS-241NS;Lane 5: 125275; Lane 6: 125434; Lane 7: 125722; Lane 8: ReferenceStandard. C3955.

FIG. 5. The primary amino acid structure of Sacrosidase consists of 513amino acids. Sacrosidase has the amino acid sequence shown below (SEQ IDNO:1).

DETAILED DESCRIPTION OF THE INVENTION

This invention provides an ultrapure hypoallergic sacrosidase that meetsthe pharmaceutical standards for human prescription use, with reducedallergenic properties. The sacrosidase is also organolepticallyimproved, since aqueous solutions thereof are odorless. Solutions ofsacrosidase that are less pure can exhibit unpleasant organolepticproperties, such as a “yeasty” smell, unappetizing color and otherodors.

Common Names: Invertase, Sacrosidase

Trade Name: Sucraid® (sacrosidase) oral solution (Drug Product)

USAN: Sacrosidase

Chemical: β-D-fructofuranoside fructohydrolase

Synonyms: β-D-fructofuranosidase

-   -   β-D-fructofuranosidase    -   β-fructofuranosidase    -   β-fructofuranoside fructohydrolase    -   β-fructopyranosidase    -   β-fructosidase    -   β-invertase    -   Fructosylinvertase    -   Invertase    -   Invertin    -   Glucosucrase    -   Saccharase    -   Sucrase    -   Sucrose hydrolase    -   Exo-β-(2,6)-Fructofuraosidase    -   Maxivert L 1000    -   Yeast Sucrase (YS)

CAS Number: 85897-35-4

-   -   9001-57-4

Summary of the Purification Process

This invention provides a chromatography free process to produceultrapure sacrosidase protein that meets pharmaceutical standards forhuman prescription use. Never before has a non-chromatographic methodbeen developed to produce such ultra-high purity protein, particularlysacrosidase, overcoming prior shortcomings due to inadequate purity. Anon-chromatographic purification process engenders significant cost andtime savings during manufacturing. Provided herein is study drugcharacterization, purity evidence, and finally clinical proof of theimproved unique hypoallergenic formulation.

The steps of a preferred embodiment of the present sacrosidase isolationand purification process are summarized in FIG. 1.

-   -   1. Digest yeast saccharomyces cerevisiae with papain in an        aqueous buffered solution at a pH of about 7.0 and at a        temperature of about 30-34° C.    -   2. The papain digestion cleaves all proteins into small        molecular weight fragments that can later be effectively        diafiltered away from the very large, papain resistant,        sacrosidase enzyme, which in solution has an apparent native        solution molecular weight of 500,000+ grains per mole (Daltons)        Sacrosidase having the extensive glycosylation produced in yeast        is resistant to papain digestion.    -   3. Use of diatomaceous earth with two different pore and/or        particle sizes is preferred to remove most of the insoluble        components, and likely other soluble and insoluble impurities.        At the chosen pH, ionic strength, and concentrations, a large        increase in purity occurs using these two steps in series that        also prepares the resulting particulate free solution for        diafiltration. Removal of insoluble components also prevents the        dialysis membrane filters in the ultrafiltration unit from        clogging. This increases the processing speed.    -   4. Diafiltration using an experimentally determined optimal pore        size filter to balance purity with yield. The present process        employs a minimum 40 kiloDalton cutoff filter to achieve an        ultrapure hypoallergenic odorless solution of sacrosidase. The        solution has a sacrosidase activity of at least about 22,000        IU/mL, preferably at least about 17,000 IU/mL. Larger pore        filters give equivalent purity, but at the cost of yield.        Smaller pore size filters do not provide sufficient final        purity. Also experimentally determined was the number of batch        volumes against which to diafilter: only 4+ diafiltration        volumes allows for purity adequate to meet the requirements for        a regulated bulk drug substance for human pharmaceutical use.    -   5. Final formulation of the drug substance with glycerol,        preferably employs sterile filtration to ensure lot bioburden        that meets FDA standards. The drug substance is stored in drums        at about −18° C. to −25° C. for long term storage, until        processed/concentration adjusted/packaged to yield the Sucraid®        drug product. At this point, the sacrosidase activity is at        least about 10,000 IU/mL.

The invention will be further described by reference to the followingdetailed examples, wherein the terms “SDS-PAGE” and “RASA” as usedherein are defined with reference to the following techniques.

Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis

Samples of the sacrosidase bulk drug substance containing predeterminedamounts of total protein, were analyzed by Charles Rivers Laboratory,Malvern, Pa. and the assays were validated by Tekagen, Inc., Malvern,Pa.

Reducing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis) is used to separate mixtures of polypeptides (in anelectric field) into distinct bands. Mobility under these conditions isprimarily determined by molecular size. Staining with ColloidalCoomassie Blue allows for visual and/or densitometric analysis of theresolved bands. This method is linear for loads from 0.25 μg to 15 μgwith a lower limit of detection of 0.1 μg per band, e.g., the limits ofdetection and quantification of sacrosidase were determined to be 0.1μg. The measured sacrosidase band area correlated linearly with theprotein load.

In the assays herein below, the sacrosidase-containing samples describedwere reduced with 2-mercaptoethanol in SDS sample buffer. Samples wereadjusted to about 1.0 mg/mL with water and treated with one volume ofthe mercaptoethanol in SDS buffer at 100® C. for 3 min. todenature/reduce the protein. Samples were run in an XCell SureLockMini-Cell® (Invitrogen) using Novex® 4·20% Tris-Glycine Gel (Invitrogen)using an SDS running buffer for 90 min. at 120 volts. Gels were stainedwith Coomassie Blue and washed in water. Gels were scanned using aPersonal Densitometer SI (Molecular Dynamics).

Papain Quantification by Enzyme-Linked Immunosorbent Assay (ELISA)

Samples of the sacrosidase bulk drug substance were dialyzed against PBSand any papain was inhibited with E-64. The sample was assayed forpapain content by Warnex Laboratories, Quebec, Canada, using aquantitative sandwich-type ELISA. A polyclonal capture antibody (rabbit)specific to papain is coated in each well of a microplate. When presentin the samples, papain binds to the capture antibody. A secondanti-papain antibody (goat) conjugated with horse-radish peroxidase(HRP) is added which binds to any captured papain. After washing, theO-phenlenediamine substrate is added with 0.03% H₂O₂ to produce anenzymatic color reaction with HRP. The reaction is stopped with asolution of sulfuric acid (stopping solution). The color intensityformed by the enzymatic reaction is measured by spectrophotometry.

The quantity of papain in the samples was calculated based on a standardcurve, where the average OD values of each of the standards are on theY-axis and the corresponding concentration of papain is on the X-axis.To determine the final concentration of papain in the samples theconcentration obtained from the standard curve is multiplied by thedilution factor. The quantity of papain is calculated automatically bythe Gen5 software. The lower limit of quantification of papain is 3.13ng/mL. The lower limit detection of papain is 1.56 ng/mL.

Example 1: Manufacturing Process for Ultrapure Sacrosidase

Deionized water (1700 kg) is charged to a 1000 gallon reactor and heatedto 32±2° C. at which time Saccharomyces cerevisiae yeast (360 kg) isadded and the slurry is mixed for 20-30 min. The pH is checked and if itis found to be below 6.9, the pH is adjusted using a 25% sodiumhydroxide solution until the pH is 7.0±0.1.

Papain (3.4 kg) 100 TU/mg is added followed by hydrogen peroxide (35%,2.8 kg). The slurry is stirred at 32±2° C. for the next 17.5 to 20 h;after every 3.5-4 h the is adjusted to 7.0±0.1 and hydrogen peroxide(34%, 2.8 kg each portion, 11.2 kg total) is added.

Once the 17.5-20 hours of mixing is complete the mixture is cooled tobetween 22° C. and 26° C. at which point 25 kg of 85% phosphoric acid isadded over a 20-30 min period. The pH of the slurry is checked and thepH is further adjusted to 4.0±0.1 by addition of 85% phosphoric acid.

Diatomaceous earth (Celpure® S1000, 210 kg) is added to the mixturefollowed by an additional 700 kg of deionized water and the slurry ismixed for 30-40 min. The slurry is filtered in four equal portionsthrough a sparkler filter to remove the cell debris and any other solidsfrom the product in solution. For each portion of the filtration, oncethe filtrate collection rate stops the filter bundle is washed withfresh deionized water (400 kg). These washes are combined with theinitial filtrates.

After the fourth filtration is complete the combined filtrates andwashes are returned to the 1000-gallon reactor and treated with hydrogenperoxide (34%, 2.0 kg). The solution is decolorized by treatment withactivated carbon (20×40 mesh, 60 kg) and aluminum sulfate (9.4 kg); theresulting slurry is mixed for 1.5-2 h.

Diatomaceous earth (Celpure® S100, 32 kg) is added and mixed for anadditional 30 min. The solids are removed by filtration through thesparkler filter. The reactor is cleaned to remove any residual solidsthat may be present and the filtrate from decolorization is returned tothe reactor through a 0.2-micron in-line filter. The solution is thenconcentrated to about 250 kg using an ultrafiltration unit that has beenequipped with 40 kilo-Dalton cutoff filters.

The concentrated sacrosidase solution is then purified by diafiltrationagainst at least four volumes (˜1000 L) of citric acid buffer solution.This buffer solution is prepared from deionized water (1000 kg), citricacid (9.2 kg), sodium citrate (11.2 kg) and the pH of the solution isadjusted to 4.6±0.1 by addition of 25% sodium hydroxide. Additionalvolumes of buffer can be used to increase the overall protein puritywhile continuing to reduce residual levels of impurities in the finalformulated solution. Typically four volumes (˜1000 L) of citric acidbuffer has been sufficient during diafiltration to purify thesacrosidase protein retained in the solution to >99% purity and toachieve papain levels <50 ppb.

Once the diafiltration is complete the solution is further concentrateduntil the material has an assay value ≥26,000 IU/mL in the sacrosidaseassay test. The pH of the solution is then checked and adjusted to4.3±0.1 by either use of 25% sodium hydroxide solution if the pH is<4.15 or by use of 85% phosphoric acid if the pH of the solution is>4.45.

Once the pH adjustment is complete the solution is transferred to amixing vessel and an about equal weight of glycerin is added to producethe sacrosidase drug substance. The resulting water-glycerin solution ismixed for 10 min. The target drug substance has >10,000 IU/mL ofsacrosidase and about 45-54 wt-% glycerol. Once the mixing is completethe solution is transferred to drums through two 0.2 micron nylonin-line filters that are set up in series. The drummed solution or “drugsubstance” is stored at room temperature for 8-36 h prior to being movedinto long-term storage at about −20° C. Portions can be removed asneeded and diluted and packaged to yield the Sucraid® drug product.

Example 2: Coomassie Blue Stained Reduced SDS-PAGE Gel withDensitometry—Optimal Load

The gel shown in FIG. 2 is a Coomassie stained, reduced SDS-PAGEcomparing 3 lots of a prior sacrosidase manufacturer (Lanes 3, 4, 5)that was manufactured with an older manufacturing process, with 3 lotsof sacrosidase (Lanes 6, 7, 8) manufactured using the new ultra-purehypoallergenic manufacturing process. Reference standard C4546 is thereference standard currently in use and was produced from the olderprocess. Reference standard C3955 is an older-yet reference standardfrom the original Sacrosidase manufacturer.

The densitometry scan of this optimally loaded SDS-PAGE gel is shownbelow (Table 1). These results clearly show that the sacrosidasemanufactured using the new process is ultrapure with no detectableprotein impurities. The cGMP validation of this method established alimit of detection of less than 0.1 micrograms.

TABLE 1 Lot Lane Band Band Sample Type Number No. No. Volume % DensityReference C4546 2. 1. 1823 94.31 Standard 2. 2. 110 5.69 Old ProcessSTS- 3. 1. 2376 94.93 199NS 3. 2. 127 5.07 Old Process STS- 4. 1. 282294.83 220NS 4. 2. 154 5.17 Old Process STS- 5. 1. 2590 94.46 241NS 5. 2.152 5.54 New Process 125275 6. 1. 2356 >99.9 New Process 125434 7. 1.2331 >99.9 New Process 125722 8. 1. 2159 >99.9 Reference C3955 9. 1.2256 >99.9 Standard* *Purified by chromatography. Experimental sampleonly.

Example 3: Coomassie Stained Reduced SDS-PAGE Gel withDensitometry—Overloaded Gel

In a second protocol reduced SDS-PAGE gels were overloaded with 20 μg ofprotein (FIG. 3). In the Coomassie stained gels, a small impurity bandis visible in the present sacrosidase drug substance, but the absolutepurity of the sacrosidase of the invention (˜97%, Lanes 6, 7, 8) isstill superior to the older sacrosidase (˜90%, Lanes 3, 4, 5). Table 2summarizes the information from this gel.

TABLE 2 Protein Sample Sample Qty Lane Band Band Band Band % Type Lot(μg) No. No. rf Volume Sum Density Reference C4546 20 2. 1. 0.151 44194867 90.80 Standard 2. 2. 0.561 448 9.20 Old STS- 20 3. 1. 0.145 50635588 90.60 Process 199NS 3. 2. 0.568 525 9.40 Old STS- 20 4. 1. 0.1325460 5969 91.47 Process 220NS 4. 2. 0.559 509 8.53 Old STS- 15 5. 1.0.132 4577 5003 91.49 Process 241NS 5. 2. 0.550 426 8.51 New 125275 156. 1. 0.126 5157 5292 97.45 Process 6. 2. 0.542 135 2.55 New 125434 157. 1. 0.134 4820 4936 97.65 Process 7. 2. 0.546 116 2.35 New 125722 158. 1. 0.128 4832 4931 97.99 Process 8. 2. 0.550 99 2.01 Reference C395515 9. 1. 0.257 5640 6326 89.16 Standard 9. 2. 0.450 69 1.09 9. 3. 0.490135 2.13 9. 4. 0.554 108 1.71 9. 5. 0.609 96 1.52 9. 6. 0.669 143 2.269. 7. 0.732 135 2.13

Example 4: Optimal and Overloaded Native Gels

In a third protocol non-reduced samples were run on native PAGE gels.Both optimal (5 μg protein) and overloaded (20, 30 & 60 μg protein)protein were applied to polyacrylamide gels then stained with CoomassieBlue.

FIG. 4 depicts an overloaded (15 μg) Coomassie stained native gel.Densitometry scanning of the gel show purity values of 97.59, 99.02, and99.31 for sacrosidase made by the older sacrosidase process and 100%,100% and 100% for the sacrosidase manufactured using the presentultrapure process. This gel clearly demonstrates the ultrapure qualityof the sacrosidase manufactured using the new process. Table 3summarizes the information from this gel.

TABLE 3 Protein Sample Sample Qty Lane Band Band Band Band % Type Lot(μg) No. No. rf Volume Sum Density Reference C4546 15 1. 1. 0.170 1003410167 98.69 1. 2. 0.492 62 0.61 1. 3. 0.711 71 0.70 Old STS- 15 2. 1.0.115 8926 9146 97.59 Process 199NS 2. 2. 0.495 51 0.56 2. 3. 0.689 860.94 2. 4. 0.896 83 0.91 Old STS- 15 3. 1. 0.120 8473 8557 99.02 Process220NS 3. 2. 0.679 84 0.98 Old STS- 15 4. 1. 0.150 8670 8730 99.31Process 241NS 4. 2. 0.662 60 0.69 New 125275 15 5. 1. 0.166 9100 9100100 Process New 125434 15 6. 1. 0.129 7967 7967 100 Process New 12572215 7. 1. 0.141 8605 8605 100 Process Reference C3955 15 8. 1. 0.19013693 13744 99.63 8. 2. 0.573 51 0.37

Example 5: Three Order of Magnitude (1,000 Fold) Reduction in a ProteinImpurity

The FDA recalled all topical papain products in 2008 due to risk ofhypersensitivity reactions(www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2008/ucm116956.htm)and a REMS was required for sacrosidase that same year(www.fda.gov/downloads/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/UCM144251.pdf). The present process demonstrates animpressive three order of magnitude reduction in the papain levels ofthe drug substance to nearly undetectable levels with a very sensitiveELISA (LOQ of 3 ng/mL) using this new chromatography-free process. LOQis the limit of quantitation. These lots below were the end result ofthree process validation lots from the present process (Lots 125275,125897, 125907) versus the old manufacturer lots (STS-199NS, STS-220NS,STS-241NS) which carried an FDA imposed papain limit of 10,000 ng/mL.

TABLE 4 Sacrosidase lot number with papain concentration underneath inng/mL STS- STS- STS- 199NS 220NS 241NS 125275 125897 125907 7300 80505470 <3 <3 <3

This example shows the substantial level of reduction of papain toundetectable levels. Papain at 23 kilo-Daltons is a key marker proteinthat is likely representative of any other proteins below the 40 KDcutoff limit of the ultrafiltration filter used, along with the proteinscleaved by papain that fall above the 40 KD molecular weight cut-off,with the exception of sacrosidase which is resistant to papain cleavagein its three dimensional native form in solution.

Example 6: Organoleptic Properties Improved

While diafiltering the solution after concentration, an operator wastasked with observing the color and smell of the concentratedsacrosidase solution. As the number of wash volumes increased from no(zero) diafiltration wash volumes up to four (4) diafiltration washvolumes, the operator observed that all yeast smell was removed from thesacrosidase solution and the yellow color was dramatically reduced toalmost no yellow color. These organoleptic properties of this newultrapure formulation are important for young pediatric patientcompliance as the “yeasty smell” is not tolerated well by some youngpatients leading to poor compliance with their prescribed Sucraiddosing.

Example 7: Clinical Evidence of Improved Safety and Continued Efficacy

A 5 year old female CSID patient was first dosed with the oldformulation of Sucraid in August 2012, and as instructed by the FDAapproved labeling (package insert) due to the possibility of allergicreactions, she was dosed the first time within her physicians' offices.This patient manifested a whole body case of hives immediately afterdosing with the old formulation of Sucraid. Further dosing with Sucraidwas therefore precluded. The family was eager to find a solution totheir daughters gastrointestinal problems, so acceded to a secondchallenge with different lot of the old formulation of Sucraid about 2months later. Again, hives manifested, and so this patient waspermanently precluded from using the commercially marketed FDA approvedSucraid to treat her genetically determined sucrase deficiency.

After this new ultrapure hypoallergenic formulation of sacrosidase wasdeveloped, an IND was filed and authorized by FDA, along withInstitutional Review Board (IRB) approval at the Mayo Clinic inRochester Minn. for a test dosing protocol in this same patient inNovember 2013. After treatment with the new ultrapure hypoallergenicformulation of sacrosidase, this 5 year old patient no longer manifestedallergic reactions or symptoms, including hives. Further, after takingthe new ultrapure hypoallergenic formulation of Sucraid her CSIDsymptoms were completely ameliorated, including normalizing to 1-2formed stools per day versus >6+ watery diarrhea type bowel movementsper day, elimination of her abdominal pain, and elimination of herabdominal distension, so that this pediatric patient is now able toutilize this life changing medication to treat her disease. The newultrapure hypoallergenic formulation of sacrosidase is being provided tothis patient for chronic use pursuant to an FDA approved IND along withMayo IRB approval with excellent results for control of her CSIDdisease.

What is claimed is:
 1. A method for treating a subject who lacksendogenous sucrase activity, comprising orally administering aneffective amount of a protein composition consisting essentiallysacrosidase having a band volume ratio of sacrosidase to other proteinscomprising papain of at least about 35:1 by SDS-PAGE of about 20 □g ofsaid composition, wherein the primary structure of the sacrosidase is a513 amino acid polypeptide that is gycosylated.
 2. The method of claim 1wherein the band volume ratio is about 35-55:1.
 3. A method of treatinga subject who lacks endogenous sucrase activity comprising orallyadministering an effective amount of a protein composition consistingessentially of sacrosidase derived from Saccharomyces having residualpapain in a concentration of less than about 10 ng/mL papain.
 4. Themethod of claim 3 wherein the protein composition is administered in asolution.
 5. The method of claim 4 wherein the sacrosidase has an enzymeactivity of at least about 7500 IU/ml.
 6. The method of claim 4 whereina daily dose of the solution of about 2-10 mL per day does not induce anallergic reaction in a human patient afflicted with congenitalsucrase-isomaltase deficiency.
 7. The method of claim 3 wherein thesacrosidase contains less than about 3.0 ng/mL papain.
 8. The method ofclaim 3 wherein the sacrosidase contains no detectable papain by anenzyme-linked immunosorbant assay having a lower limit of quantificationof 3 ng/mL.
 9. The method of claim 3 wherein the solution of sacrosidasecontains no detectable papain by SDS-PAGE of up to about 15 □g of saidsolution.
 10. The method of claim 5 wherein the solution of sacrosidasehas enzymatic activity of about 7500-10,000 IU/ml.